24 1 General Aspects of Medical Microbiology
Defects in Immune Defenses
1
Hosts with defects in their specific and/or nonspecific immune defenses are
prone to infection.
& Primary defects. Congenital defects in the complement-dependent pha-
gocytosis system are rare, as are B and T lymphocyte defects.
& Secondary defects. Such effects are acquired, and they are much more
frequent. Examples include malnutrition, very old and very young hosts, me-
tabolic disturbances (diabetes, alcoholism), autoimmune diseases, malignan-
cies (above all lymphomas and leukemias), immune system infections (HIV),
severe primary diseases of parenchymatous organs, injury of skin or mucosa,
immunosuppressive therapy with corticosteroids, cytostatics and immuno-
suppressants, and radiotherapy.
One result of progress in modern medicine is that increasing numbers of pa-
tients with secondary immune defects are now receiving hospital treatment.
Such “problem patients” are frequently infected by opportunistic bacteria
that would not present a serious threat to normal immune defenses. Often,
the pathogens involved (“problem bacteria”) have developed a resistance to
numerous antibiotics, resulting in difficult courses of antibiotic treatment in
this patient category.
Normal Flora
Commensals (see Table 1.3, p. 9) are regularly found in certain human micro-
biotopes. The normal human microflora is thus the totality of these commen-
sals. Table 1.7 lists the most important microorganisms of the normal flora
with their localizations.
Bacteria are the predominant component of the normal flora. They
proliferate in varied profusion on the mucosa and most particularly in the
gastrointestinal tract, where over 400 different species have been counted
to date. The count of bacteria per gram of intestinal content is 101–105 in
the duodenum, 103–107 in the small intestine, and 1010–1012 in the colon.
Over 99 % of the normal mucosal flora are obligate anaerobes, dominated
by the Gram-neg. anaerobes. Although life is possible without normal flora
(e.g., pathogen-free experimental animals), commensals certainly benefit
their hosts. One way they do so is when organisms of the normal flora
manage to penetrate into the host through microtraumas, resulting in a
continuous stimulation of the immune system. Commensals also compete
for living space with overtly pathogenic species, a function known as colo-
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General Epidemiology 25
Table 1.7 Normal Microbial Flora in Humans 1
Microorganisms Microbiotopes
Skin Oral Intes- Upper re- Genital
cavity tine spiratory tract
Staphylococci +++ tract
Enterococci + + +
a-hemolytic streptococci ++ ++ ++
Anaerobic cocci ++ +++ + +
Pneumococci (+) + +
Apathogenic neisseriae (+) + ++
Apathogenic corynebacteria + + +
Aerobic spore-forming bacteria ++ +
Clostridia +
Actinomycetes ++
Enterobacteriaceae ++
Pseudomonas
Haemophilus +++ (+)
Gram-neg. anaerobes +
Spirochetes +++ +
Mycoplasmas
Fungi (yeast) (+) +++ (+) (+)
Entamoeba, Giardia, Trichomonas +++
+ (+)
++
+ ++ +
+++ +++ +++
++ +
++ + +
+++
++
+++ = numerous, ++ = frequent, + = moderately frequent, (+) = occasional occurrence
nization resistance. On the other hand, a potentially harmful effect of the
normal flora is that they can also cause infections in immunocompromised
individuals.
General Epidemiology
& Within the context of medical microbiology, epidemiology is the study of
the occurrence, causality, and prevention of infectious diseases in the popu-
lace. Infectious diseases occur either sporadically, in epidemics or pandemics,
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26 1 General Aspects of Medical Microbiology
or in endemic forms, depending on the time and place of their occurrence.
1 The frequency of their occurrence (morbidity) is described as their incidence
and prevalence. The term mortality is used to describe how many deaths are
caused by a given disease in a given population. Lethality is a measure of how
life-threatening an infection is. The most important sources of infection are
infected persons and carriers. Pathogens are transmitted from these sources
to susceptible persons either directly (person-to-person) or indirectly via in-
ert objects or biological vectors. Control of infectious diseases within a pop-
ulace must be supported by effective legislation that regulates mandatory
reporting where required. Further measures must be implemented to pre-
vent exposure, for example isolation, quarantine, disinfection, sterilization,
use of insecticides, and dispositional prophylaxis (active and passive immu-
nization, chemoprophylaxis). &
Epidemiological Terminology
Epidemiology investigates the distribution of diseases, their physiological
variables and social consequences in human populations, and the factors
that influence disease distribution (World Health Organization [WHO] defi-
nition). The field covered by this discipline can thus be defined as medical
problems involving large collectives. The rule of thumb on infectious diseases
is that their characteristic spread depends on the virulence of the pathogen
involved, the susceptibility of the threatened host species population, and
environmental factors. Table 1.8 provides brief definitions of the most impor-
tant epidemiological terms.
Transmission, Sources of Infection
Transmission
Pathogens can be transmitted from a source of infection by direct contact or
indirectly. Table 1.9 lists the different direct and indirect transmission path-
ways of pathogenic microorganisms.
Person-to-person transmission constitutes a homologous chain of infec-
tion. The infections involved are called anthroponoses. In cases in which the
pathogen is transmitted to humans from other vertebrates (and occasionally
the other way around) we have a heterologous chain of infection and the
infections are known as zoonoses (WHO definition) (Table 1.10).
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General Epidemiology 27
Table 1.8 Epidemiological Terminology 1
Term Definition
Sporadic occurrence Isolated occurrence of an infectious disease with no appar-
Endemic occurrence ent connections between localities or times of occurrence
Epidemic occurrence
Pandemic occurrence Regular and continuing occurrence of infectious diseases in
populations with no time limit
Morbidity
Incidence Significantly increased occurrence of an infectious disease
Prevalence within given localities and time periods
Mortality Significantly increased occurrence of an infectious disease
Lethality within a given time period but without restriction to given
Manifestation index localities
Incubation period
Prepatency Number of cases of a disease within a given population
(e.g., per 1000, 10 000 or 100 000 inhabitants)
Number of new cases of a disease within a given time peri-
od
Number of cases of a disease at a given point in time
(sampling date)
Number of deaths due to a disease within a given popula-
tion
Number of deaths due to a disease in relation to total
number of cases of the disease
Number of manifest cases of a disease in relation to num-
ber of infections
Time from infection until occurrence of initial disease
symptoms
Time between infection and first appearance of products of
sexual reproduction of the pathogen (e.g., worm eggs in
stool)
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28 1 General Aspects of Medical Microbiology
Tab. 1.9 Transmission Pathways of Pathogenic Microorganisms
1
Direct transmission Indirect transmission
Fecal-oral (smear infection) Transmission via food
Transmission via drinking water
Aerogenic transmission
(droplet infection) Transmission via contaminated inanimate objects
or liquids
Genital transmission Transmission via vectors (arthropods)
(during sexual intercourse) Transmission via other persons (e.g., via the hands
of hospital medical staff)
Transmission via skin (rare)
Diaplacental transmission
Perinatal transmission
(in the course of birth)
Tab. 1.10 Examples of Zoonoses Caused by Viruses, Bacteria, Protozoans, Hel-
minths, and Arthropods
Zoonoses Pathogen Reservoir hosts Transmission
Rhabdoviridae
Viral zoonoses Numerous animal Bite of diseased animals
Rabies species Ticks
Tickborne ence- Flaviviridae Wild animals
phalitis (TBE)
Bacterial zoonoses
Brucellosis Brucella spp. Cattle, pig, goat, Contact with tissues or
sheep, (dog) secretions from diseased
Lyme disease Borrelia animals; milk and dairy
burgdorferi Wild rodents; products
Plague Yersinia pestis red deer, roe deer
Rodents Ticks
Q fever Coxiella burnetii
Sheep, goat, Contact with diseased
Enteric Salmonella cattle animals; bite of rat flea
salmonellosis enterica (enteric Pig, cattle,
serovars) poultry Dust; possibly milk or
dairy products
Meat, milk, eggs
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General Epidemiology 29
Tab. 1.10 Continued: Examples of Zoonoses 1
Zoonoses Pathogen Reservoir hosts Transmission
Protozoan zoonoses Domestic cat, Postnatal toxoplasmosis:
Toxoplasmosis Toxoplasma sheep, pigs, other oral; prenatal toxoplas-
slaughter animals mosis: diaplacental
gondii
Cattle (calves), Ingestion of oocysts
Cryptosporidiosis Cryptosporidium domestic animals
hominis;
C. parvum
Helminthic zoonoses Dog, wild canines, Ingestion of eggs
Echinococcosis Echinococcus fox
granulosus,
Echinococcus
multilocularis
Taeniosis Taenia saginata, Cattle, buffalo, Ingestion of metaces-
Taenia solium, pigs todes with meat
Taenia asiatica Pigs, cattle, goat
Zoonoses caused by arthropods Dog, cat, guinea Contact with diseased
Pseudo scabies Sarcoptes spp.; pig, domestic animals
ruminants, pig
mite species
from domestic
animals
Other Zoonoses
(For details see the corresponding chapters)
Viral zoonoses Hantavirus and other bunyavirus infections; infections by
alphavirus, flavivirus, and arenavirus.
Bacterial zoonoses Ehrlichiosis; erysipeloid; campylobacteriosis; cat scratch
disease; leptospirosis; anthrax; ornithosis; rat-bite fever;
rickettsioses (variety of types); tularemia; gastroenteritis
caused by Vibrio parahaemolyticus; gastroenteritis caused
by Yersinia enterocolitica.
Protozoan zoonoses African trypanosomosis (sleeping sickness); American
trypanosomosis (Chagas disease); babesiosis; balanti-
dosis; cryptosporidosis; giardiosis; leishmaniosis; micro-
sporidosis; sarcocystosis; toxoplasmosis. "
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30 1 General Aspects of Medical Microbiology
1 Continued: Other Zoonoses
Helminthic zoonoses Cercarial dermatitis; clonorchiosis; cysticercosis; dicro-
coeliosis; diphyllobothriosis; echinococcosis; fasciolosis;
hymenolepiosis; larva migrans interna; opisthorchiosis;
paragonimosis; schistosomosis (bilharziosis); taeniosis;
toxocariosis; trichinellosis.
Zoonoses caused Flea infestation; larva migrans externa; mite infestation;
by arthropods sand flea infestation.
Sources of Infection
Every infection has a source (Table 1.11). The primary source of infection is
defined as the location at which the pathogen is present and reproduces. Sec-
ondary sources of infection are inanimate objects, materials, or third per-
sons contributing to transmission of pathogens from the primary source to
disposed persons.
Table 1.11 Primary Sources of Infection
Source of infection Explanation
Infected person The most important source; as a rule, pathogens are
excreted by the organ system through which the infection
entered; there are some exceptions
Carriers during incuba- Excretion during incubation period; typical of many viral
tion diseases
Carriers in convales- Excretion after the disease has been overcome; typical of
cence enteric salmonelloses
Chronic carriers Continued excretion for three or more months (even years)
after disease has been overcome; typical of typhoid fever
Asymptomatic carriers They carry pathogenic germs on skin or mucosa without
developing “infection”
Animal carriers Diseased or healthy animals that excrete pathogenic germs
Environment Soil, plants, water; primary source of microorganisms with
natural habitat in these biotopes
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General Epidemiology 31 1
The Fight against Infectious Diseases
Legislation
Confronting and preventing infectious diseases can sometimes involve sub-
stantial incursions into the private sphere of those involved as well as eco-
nomic consequences. For these reasons, such measures must be based on ef-
fective disease control legislation. In principle, these laws are similar in most
countries, although the details vary.
The centerpiece of every disease prevention system is provision for re-
porting outbreaks. Basically, reporting is initiated at the periphery (individual
patients) and moves toward the center of the system. Urgency level classifi-
cations of infections and laboratory findings are decided on by regional
health centers, which are in turn required to report some diseases to the
WHO to obtain a global picture within the shortest possible time.
Concrete countermeasures in the face of an epidemic take the form of pro-
phylactic measures aimed at interrupting the chain of infection.
Exposure Prophylaxis
Exposure prophylaxis begins with isolation of the source of infection, in par-
ticular of infected persons, as required for the disease at hand. Quarantine
refers to a special form of isolation of healthy first-degree contact persons.
These are persons who have been in contact with a source of infection.
The quarantine period is equivalent to the incubation period of the infectious
disease in question (see International Health Regulations, www.who.int/en/).
Further measures of exposure prophylaxis include disinfection and steri-
lization, use of insecticides and pesticides, and eradication of animal carriers.
Immunization Prophylaxis
Active immunization. In active immunization, the immune system is stimu-
lated by administration of vaccines to develop a disease-specific immunity.
Table 1.12 lists the vaccine groups used in active immunization. Table 1.13
shows as an example the vaccination schedule recommended by the Advisory
Committee on Immunization Practices of the USA (www.cdc.gov/nip). Re-
commended adult immunization schedules by age group and by medical
conditions are also available in the National Immunization Program Website
mentioned above. The vaccination calendars used in other countries deviate
from these proposals in some details. For instance, routine varicella and
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32 1 General Aspects of Medical Microbiology
Table 1.12 Vaccine Groups Used in Active Immunization
1
Vaccine group Remarks
Killed pathogens Vaccination protection often not optimum, vaccination
Living pathogens with has to be repeated several times
reduced virulence
(attenuated) Optimum vaccination protection; a single application
often suffices, since the microorganisms reproduce in
Purified microbial the vaccinated person, providing very good stimulation
immunogens of the immune system; do not use in immunocompro-
– Proteins mised persons and during pregnancy (some exceptions)
– Polysaccharides Often recombinant antigens, i.e., genetically engineered
proteins; well-known example: hepatitis B surface (HBs)
– Conjugate vaccines antigen
Toxoids Chemically purified capsular polysaccharides of pneu-
Experimental vaccines mococci, meningococci, and Haemophilus influenzae se-
rotype b; problem: these are T cell-independent antigens
that do not stimulate antibody production in children
younger than two years of age
Coupling of bacterial capsular polysaccharide epitopes to
proteins, e.g., to tetanus toxoid, diphtheria toxoid, or
proteins of the outer membranes of meningococci; chil-
dren between the ages of two months and two years
can also be vaccinated against polysaccharide epitopes
Bacterial toxins detoxified by formaldehyde treatment
that still retain their immunogen function
DNA vaccines. Purified DNA that codes for the viral
antigens (proteins) and is integrated in plasmid DNA or
nonreplicating viral vector DNA. The vector must have
genetic elements—for example a transcriptional promo-
ter and RNA-processing elements—that enable expres-
sion of the insert in the cells of various tissues (epider-
mis, muscle cells)
Anti-idiotype-specific monoclonal antibodies
Vaccinia viruses as carriers of foreign genes that code for
immunogens
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General Epidemiology 33 1
Table 1.13 Recommended Childhood and Adolescent Immunization Schedule—
United States, 2004
1. Hepatitis B vaccine (HepB).
Infants born to HBs-Ag-positive mothers should receive HepB and 0.5 ml HepB
Immune Globulin within 12 h of birth at separate sites.
2. Diphtheria (D) and tetanus (T) toxoids and acellular pertussis (aP) vaccine (DTaP).
The term “d” refers to a reduced dose of diphtheria toxoid.
3. Haemophilus influenzae type b conjugate vaccine (see Table 1.12).
4. Measles, mumps, and rubella vaccine (MMR).
Attenuated virus strains.
5. Varicella vaccine.
Varicella vaccine is recommended for children who lack a reliable history of
chickenpox.
6. Pneumococcal vaccine.
The heptavalent conjugate vaccine (PCV) is recommended for all children age
2–23 months. Pneumococcal polysaccharide vaccine (PPV) can be used in elder
children.
7. Hepatitis A vaccine.
The “killed virus vaccine” is recommended in selected regions and for certain
high-risk groups. Two doses should be administered at least six months apart.
8. Influenza vaccine.
Influenza vaccine is recommended annually for children with certain risk factors
(for instance asthma, cardiac disease, sickle cell disease, HIV, diabetes etc.). Chil-
dren aged –< eight years who are receiving influenza vaccine for the first time
should receive two doses separated at least four weeks.
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34 1 General Aspects of Medical Microbiology
pneumococcal vaccinations are not obligatory in Germany, Austria, and Swit-
1 zerland (see www.rki.de). To simplify the application of vaccines, licensed
combination vaccines may be used whenever any components of the com-
bination are indicated and the vaccine’s other components are not contrain-
dicated. Providers should consult the manufacturers’ inserts for detailed in-
formation.
Passive immunization. This vaccination method involves administration of
antibodies produced in a different host. In most cases, homologous (human)
hyperimmune sera (obtained from convalescent patients or patients with
multiple vaccinations) are used. The passive immunity obtained by this
method is limited to a few weeks (or months at most).
Principles of Sterilization and Disinfection
& Sterilization is defined as the killing or removal of all microorganisms and
viruses from an object or product. Disinfection means rendering an object,
the hands or skin free of pathogens. The term asepsis covers all measures
aiming to prevent contamination of objects or wounds. Disinfection and ster-
ilization makes use of both physical and chemical agents. The killing of mi-
croorganisms with these agents is exponential. A measure of the efficacy of
this process is the D value (decimal reduction time), which expresses the time
required to reduce the organism count by 90 %. The sterilization agents of
choice are hot air (180 8C, 30 minutes; 160 8C, 120 minutes) or saturated water
vapor (121 8C, 15 minutes, 2.02 Â 105 Pa; 134 8C, three minutes, 3.03 Â 105 Pa).
Gamma rays or high-energy electrons are used in radiosterilization at a re-
commended dose level of 2.5 Â 104 Gy.
Disinfection is usually done with chemical agents, the most important of
which are aldehydes (formaldehyde), alcohols, phenols, halogens (I, Cl),
and surfactants (detergents). &
Terms and General Introduction
Terms
Sterilization is the killing of all microorganisms and viruses or their complete
elimination from a material with the highest possible level of certainty.
An object that has been subjected to a sterilization process, then packaged
so as to be contamination-proof, is considered sterile.
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Principles of Sterilization and Disinfection 35 1
Killing of Prions and Thermophilic Archaea
The standard sterilization methods used in medical applications (see below) are
capable of causing irreversible damage to medically relevant microorganisms
such as bacteria, protozoans, fungi, and helminths including worm eggs. Much
more extreme processes are required to inactivate prions, such as autoclaving
at 121 8C for 4.5 hours or at 134 8C for 30 minutes. Hyperthermophilic archaea
forms have also been discovered in recent years (see p. 5) that proliferate at tem-
peratures of 100 8C and higher and can tolerate autoclaving at 121 8C for one hour.
These extreme life forms, along with prions, are not covered by the standard defini-
tions of sterilization and sterility.
Disinfection is a specifically targeted antimicrobial treatment with the ob-
jective of preventing transmission of certain microorganisms. The purpose of
the disinfection procedure is to render an object incapable of spreading in-
fection.
Preservation is a general term for measures taken to prevent microbe-
caused spoilage of susceptible products (pharmaceuticals, foods).
Decontamination is the removal or count reduction of microorganisms
contaminating an object.
The objective of aseptic measures and techniques is to prevent microbial
contamination of materials or wounds.
In antiseptic measures, chemical agents are used to fight pathogens in or
on living tissue, for example in a wound.
The Kinetics of Pathogen Killing
Killing microorganisms with chemical agents or by physical means involves a
first-order reaction. This implies that no pathogen-killing method kills off all
the microorganisms in the target population all at once and instantaneously.
Plotting the killing rate against exposure time in a semilog coordinate system
results in a straight-line curve (Fig. 1.7).
Sigmoid and asymptotic killing curves are exceptions to the rule of expo-
nential killing rates. The steepness of the killing curves depends on the sen-
sitivity of the microorganisms to the agent as well as on the latter’s effective-
ness. The survivor/exposure curve drops at a steeper angle when heat is ap-
plied, and at a flatter angle with ionizing radiation or chemical disinfectants.
Another contributing factor is the number of microorganisms contaminating
a product (i.e., its bioburden): when applied to higher organism concentra-
tions, an antimicrobial agent will require a longer exposure time to achieve
the same killing effect.
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36 1 General Aspects of Medical Microbiology
Bacterial Death Kinetics
1
Surviving cell count in log units Bacteria species A Fig.1.7 The death rate varies
Bacteria species B among bacterial species. The
higher the initial concentration
of a bacterial culture, the longer
an applied antimicrobial agent
will require to achieve the same
effect.
Time exposed to antimicrobial agent
Standard sterilization methods extend beyond killing all microorganisms
on the target objects to project a theoretical reduction of risk, i.e., the number
of organisms per sterilized unit should be equal to or less than 10–6.
The D value (decimal reduction time), which expresses the time required
to reduce the organism count by 90 %, is a handy index for killing effective-
ness.
The concentration (c) of chemical agents plays a significant role in patho-
gen-killing kinetics. The relation between exposure time (t) and c is called the
dilution coefficient (n): t Á cn = constant. Each agent has a characteristic coef-
ficient n, for instance five for phenol, which means when c is halved the ex-
posure time must be increased by a factor of 32 to achieve the same effect.
The temperature coefficient describes the influence of temperature on the
effectiveness of chemical agents. The higher the temperature, the stronger
the effect, i.e., the exposure time required to achieve the same effect is re-
duced. The coefficient of temperature must be determined experimentally
for each combination of antimicrobial agent and pathogen species.
Mechanisms of Action
When microorganisms are killed by heat, their proteins (enzymes) are irre-
versibly denatured. Ionizing radiation results in the formation of reactive
groups that contribute to chemical reactions affecting DNA and proteins. Ex-
posure to UV light results in structural changes in DNA (thymine dimers) that
prevent it from replicating. This damage can be repaired to a certain extent
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Principles of Sterilization and Disinfection 37 1
by light (photoreactivation). Most chemical agents (alcohols, phenols, alde-
hydes, heavy metals, oxidants) denature proteins irreversibly. Surfactant
compounds (amphoteric and cationic) attack the cytoplasmic membrane.
Acridine derivatives bind to DNA to prevent its replication and function (tran-
scription).
Physical Methods of Sterilization and Disinfection
Heat
The application of heat is a simple, cheap and effective method of killing
pathogens. Methods of heat application vary according to the specific appli-
cation.
& Pasteurization. This is the antimicrobial treatment used for foods in li-
quid form (milk):
— Low-temperature pasteurization: 61.5 8C, 30 minutes; 71 8C, 15 seconds.
— High-temperature pasteurization: brief (seconds) of exposure to 80–85 8C
in continuous operation.
— Uperization: heating to 150 8C for 2.5 seconds in a pressurized container
using steam injection.
& Disinfection. Application of temperatures below what would be required
for sterilization. Important: boiling medical instruments, needles, syringes,
etc. does not constitute sterilization! Many bacterial spores are not killed
by this method.
& Dry heat sterilization. The guideline values for hot-air sterilizers are as
follows: 180 8C for 30 minutes, 160 8C for 120 minutes, whereby the objects to
be sterilized must themselves reach these temperatures for the entire pre-
scribed period.
& Moist heat sterilization. Autoclaves charged with saturated, pressurized
steam are used for this purpose:
— 121 8C, 15 minutes, one atmosphere of pressure (total: 202 kPa).
— 134 8C, three minutes, two atmospheres of pressure (total: 303 kPa).
In practical operation, the heating and equalibriating heatup and equalizing
times must be added to these, i.e., the time required for the temperature in
the most inaccessible part of the item(s) to be sterilized to reach sterilization
level. When sterilizing liquids, a cooling time is also required to avoid boiling
point retardation.
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38 1 General Aspects of Medical Microbiology
The significant heat energy content of steam, which is transferred to the
1 cooler sterilization items when the steam condenses on them, explains why it
is such an effective pathogen killer. In addition, the proteins of microorgan-
isms are much more readily denatured in a moist environment than under
dry conditions.
Radiation
& Nonionizing radiation. Ultra-violet (UV) rays (280–200 nm) are a type of
nonionizing radiation that is rapidly absorbed by a variety of materials. UV
rays are therefore used only to reduce airborne pathogen counts (surgical
theaters, filling equipment) and for disinfection of smooth surfaces.
& Ionizing radiation. Two types are used:
— Gamma radiation consists of electromagnetic waves produced by nuclear
disintegration (e.g., of radioisotope 60Co).
— Corpuscular radiation consists of electrons produced in generators and
accelerated to raise their energy level.
Radiosterilization equipment is expensive. On a large scale, such systems are
used only to sterilize bandages, suture material, plastic medical items, and
heat-sensitive pharmaceuticals. The required dose depends on the level of
product contamination (bioburden) and on how sensitive the contaminating
microbes are to the radiation. As a rule, a dose of 2.5 Â 104 Gy (Gray) is con-
sidered sufficient.
One Gy is defined as absorption of the energy quantum one joule (J)
per kg.
Filtration
Liquids and gases can also be sterilized by filtration. Most of the available
filters catch only bacteria and fungi, but with ultrafine filters viruses and
even large molecules can be filtered out as well. With membrane filters, re-
tention takes place through small pores. The best-known type is the mem-
brane filter made of organic colloids (e.g., cellulose ester). These materials can
be processed to produce thin filter layers with gauged and calibrated pore
sizes. In conventional depth filters, liquids are put through a layer of fibrous
material (e.g., asbestos). The effectiveness of this type of filter is due largely
to the principle of adsorption. Because of possible toxic side effects, they are
now practically obsolete.
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Principles of Sterilization and Disinfection 39 1
Chemical Methods of Sterilization and Disinfection
Ethylene oxide. This highly reactive gas (C2H4O) is flammable, toxic, and a
strong mucosal irritant. Ethylene oxide can be used for sterilization at low
temperatures (20–60 8C). The gas has a high penetration capacity and can
even get through some plastic foils. One drawback is that this gas cannot
kill dried microorganisms and requires a relative humidity level of 40–
90 % in the sterilizing chamber. Ethylene oxide goes into solution in plastics,
rubber, and similar materials, therefore sterilized items must be allowed to
stand for a longer period to ensure complete desorption.
Aldehydes. Formaldehyde (HCHO) is the most important aldehyde. It can be
used in a special apparatus for gas sterilization. Its main use, however, is in
disinfection. Formaldehyde is a water-soluble gas. Formalin is a 35 % solution
of this gas in water. Formaldehyde irritates mucosa; skin contact may result in
inflammations or allergic eczemas. Formaldehyde is a broad-spectrum ger-
micide for bacteria, fungi, and viruses. At higher concentrations, spores
are killed as well. This substance is used to disinfect surfaces and objects
in 0.5–5 % solutions. In the past, it was commonly used in gaseous form to
disinfect the air inside rooms (5 g/m3). The mechanism of action of formal-
dehyde is based on protein denaturation.
Another aldehyde used for disinfection purposes is glutaraldehyde.
Alcohols. The types of alcohol used in disinfection are ethanol (80 %), propanol
(60 %), and isopropanol (70 %). Alcohols are quite effective against bacteria and
fungi, less so against viruses. They do not kill bacterial spores. Due to their
rapid action and good skin penetration, the main areas of application of al-
cohols are surgical and hygienic disinfection of the skin and hands. One dis-
advantage is that their effect is not long-lasting (no depot effect). Alcohols
denature proteins.
Phenols. Lister was the first to use phenol (carbolic acid) in medical applica-
tions. Today, phenol derivatives substituted with organic groups and/or halo-
gens (alkylated, arylated, and halogenated phenols), are widely used. One
common feature of phenolic substances is their weak performance against
spores and viruses. Phenols denature proteins. They bind to organic materials
to a moderate degree only, making them suitable for disinfection of excreted
materials.
Halogens. Chlorine, iodine, and derivatives of these halogens are suitable for
use as disinfectants. Chlorine and iodine show a generalized microbicidal ef-
fect and also kill spores.
Chlorine denatures proteins by binding to free amino groups; hypochlo-
rous acid (HOCl), on the other hand, is produced in aqueous solutions, then
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40 1 General Aspects of Medical Microbiology
Surfactant Disinfectants
1 R2 R3 +
N X– H Fig.1.8 Quaternary ammonium com-
RN pounds (a) and amphoteric substances
R1 R4 CH2 COOH (b) disrupt the integrity and function
ab of microbial membranes.
disintegrates into HCl and 1/2 O2 and thus acts as a powerful oxidant. Chlorine
is used to disinfect drinking water and swimming-pool water (up to 0.5 mg/l).
Calcium hypochlorite (chlorinated lime) can be used in nonspecific disinfec-
tion of excretions. Chloramines are organic chlorine compounds that split off
chlorine in aqueous solutions. They are used in cleaning and washing pro-
ducts and to disinfect excretions.
Iodine has qualities similar to those of chlorine. The most important iodine
preparations are the solutions of iodine and potassium iodide in alcohol (tinc-
ture of iodine) used to disinfect skin and small wounds. Iodophores are com-
plexes of iodine and surfactants (e.g., polyvinyl pyrrolidone). While iodo-
phores are less irritant to the skin than pure iodine, they are also less effective
as germicides.
Oxidants. This group includes ozone, hydrogen peroxide, potassium perman-
ganate, and peracetic acid. Their relevant chemical activity is based on the
splitting off of oxygen. Most are used as mild antiseptics to disinfect mucosa,
skin, or wounds.
Surfactants. These substances (also known as surface-active agents, tensides,
or detergents) include anionic, cationic, amphoteric, and nonionic detergent
compounds, of which the cationic and amphoteric types are the most effec-
tive (Fig. 1.8).
The bactericidal effect of these substances is only moderate. They have no
effect at all on tuberculosis bacteria (with the exception of amphotensides),
spores, or nonencapsulated viruses. Their efficacy is good against Gram-pos-
itive bacteria, but less so against Gram-negative rods. Their advantages in-
clude low toxicity levels, lack of odor, good skin tolerance, and a cleaning ef-
fect.
Practical Disinfection
The objective of surgical hand disinfection is to render a surgeon’s hands as
free of organisms as possible. The procedure is applied after washing the
hands thoroughly. Alcoholic preparations are best suited for this purpose,
although they are not sporicidal and have only a brief duration of action.
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Principles of Sterilization and Disinfection 41 1
Alcohols are therefore often combined with other disinfectants (e.g., quater-
nary ammonium compounds). Iodophores are also used for this purpose.
The purpose of hygienic hand disinfection is to disinfect hands contami-
nated with pathogenic organisms. Here also, alcohols are the agent of choice.
Alcohols and/or iodine compounds are suitable for disinfecting patient’s
skin in preparation for surgery and injections.
Strong-smelling agents are the logical choice for disinfection of ex-
cretions (feces, sputum, urine, etc.). It is not necessary to kill spores in
such applications. Phenolic preparations are therefore frequently used. Con-
taminated hospital sewage can also be thermally disinfected (80–100 8C)
if necessary.
Surface disinfection is an important part of hospital hygiene. A combination
of cleaning and disinfection is very effective. Suitable agents include aldehyde
and phenol derivatives combined with surfactants.
Instrument disinfection is used only for instruments that do not cause inju-
ries to skin or mucosa (e.g., dental instruments for work on hard tooth sub-
stance). The preparations used should also have a cleaning effect.
Laundry disinfection can be done by chemical means or in combination with
heat treatment. The substances used include derivatives of phenols, alde-
hydes and chlorine as well as surfactant compounds. Disinfection should pre-
ferably take place during washing.
Chlorine is the agent of choice for disinfection of drinking water and
swimming-pool water. It is easily dosed, acts quickly, and has a broad dis-
infectant range. The recommended concentration level for drinking water is
0.1–0.3 mg/l and for swimming-pool water 0.5 mg/l.
Final room disinfection is the procedure carried out after hospital care of an
infection patient is completed and is applied to a room and all of its furnish-
ings. Evaporation or atomization of formaldehyde (5 g/m3), which used to be
the preferred method, requires an exposure period of six hours. This proce-
dure is now being superseded by methods involving surface and spray dis-
infection with products containing formaldehyde.
Hospital disinfection is an important tool in the prevention of cross-infec-
tions among hospital patients. The procedure must be set out in written
form for each specific case.
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2 Basic Principles of Immunology 43
2
R. M. Zinkernagel
Introduction
& Resistance to disease is based on innate mechanisms and adaptive or
acquired immunity. Acquired immune mechanisms act in a specific manner
and function to supplement the important nonspecific or natural resistance
mechanisms such as physical barriers, granulocytes, macrophages, and
chemical barriers (lysozymes, etc.). The specific immune mechanisms
constitute a combination of less specific factors, including the activation of
macrophages, complement, and necrosis factors; the early recognition of
invading agents, by cells exhibiting a low level of specificity, (natural killer
cells, cd [gamma-delta] T cells); and systems geared toward highly specific
recognition (antibodies and ab [alpha-beta] T cells).
Many components of the specific immune defenses also contribute to
nonspecific or natural defenses such as natural antibodies, complement,
interleukins, interferons, macrophages, and natural killer cells. &
In the strict sense, “immunity” defines an acquired resistance to infectious
disease that is specific, i.e., resistance against a particular disease-causing
pathogen. For example, a person who has had measles once will not suffer
from measels a second time, and is thus called immune. However, such spe-
cific or acquired immune mechanisms do not represent the only factors
which determine resistance to infection. The canine distemper virus is a close
relative of the measles virus, but never causes an infection in humans. This
kind of resistance is innate and nonspecific. Our immune system recognizes
the pathogen as foreign based on certain surface structures, and eliminates it.
Humans are thus born with resistance against many microorganisms (innate
immunity) and can acquire resistance to others (adaptive or acquired im-
munity; Fig. 2.1). Activation of the mechanisms of innate immunity, also
known as the primary immune defenses, takes place when a pathogen
breaches the outer barriers of the body. Specific immune defense factors
are mobilized later to fortify and regulate these primary defenses. Responses
of the adaptive immune system not only engender immunity in the strict
sense, but can also contribute to pathogenic processes. The terms immuno-
pathology, autoimmunity, and allergy designate a group of immune
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44 2 Basic Principles of Immunology
The Components of Anti-Infection Defense
Innate, Physical Cellular Chemical barriers
nonspecific barriers defenses pH, lipids, enzymes,
defenses
2 Skin, mucosa Granulocytes complement factors,
Macrophages interleukins, acute
NK cells phase proteins,
antimicrobial peptides
surfactants
Acquired, Cellular Humoral
specific defenses defenses
immunity Antibodies
Lymphokines,
cytokines
Cytotoxic T cells B cells
T helper cells
Fig. 2.1 The innate immune defense system comprises nonspecific physical,
cellular, and chemical mechanisms which are distinct from the acquired immune
defense system. The latter comprises cellular (T-cell responses) and humoral (anti-
bodies) components. Specific T cells, together with antibodies, recruit non-specific
effector mechanisms to areas of antigen presence.
phenomena causing mainly pathological effects, i.e., tissue damage due to
inadequate, misguided, or excessive immune responses. However, a failed
immune response may also be caused by a number of other factors. For
instance, certain viral infections or medications can suppress or attenuate
the immune response. This condition, known as immunosuppression, can
also result from rare genetic defects causing congenital immunodeficiency.
The inability to initiate an immune response to the body’s own self anti-
gens (also termed autoantigens) is known as immunological tolerance.
Anergy is the term used to describe the phenomenon in which cells in-
volved in immune defense are present but are not functional.
An immune response is a reaction to an immunological stimulus. The
stimulating substances are known as antigens and are usually proteins or
complex carbohydrates. The steric counterparts of the antigens are the anti-
bodies, i.e., immunoreceptors formed to recognize segments, roughly 8–15
amino acids long, of the folded antigenic protein. These freely accessible
structural elements are known as epitopes when present on the antigens,
or as antigen-binding sites (ABS) from the point of view of the immuno-
receptors. Presented alone, an epitope is not sufficient to stimulate an immu-
nological response. Instead responsiveness is stimulated by epitopes con-
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The Immunological Apparatus 45 2
stituting part of a macromolecule. This is why the epitope component of an
antigen is terminologically distinguished from its macromolecular carrier;
together they form an immunogen. B lymphocytes react to the antigen sti-
mulus by producing antibodies. The T lymphocytes (T cells) responsible for
cellular immunity are also activated. These cells can only recognize protein
antigens that have been processed by host cells and presented on their sur-
face. The T-cell receptors recognize antigen fragments with a length of 8–12
sequential amino acids which are either synthesized by the cell itself or pro-
duced subsequent to phagocytosis and presented by the cellular transplan-
tation antigen molecules on the cell surface. The T cells can then complete
their main task—recognition of infected host cells—so that infection is halted.
Our understanding of the immune defense system began with studies of
infectious diseases, including the antibody responses to diphtheria, dermal
reactions to tuberculin, and serodiagnosis of syphilis. Characteriztion of
pathological antigens proved to be enormously difficult, and instead erythro-
cyte antigens, artificially synthesized chemical compounds, and other more
readily available proteins were used in experimental models for more than 60
years. Major breakthroughs in bacteriology, virology, parasitology, biochem-
istry, molecular biology, and experimental embryology in the past 30–40
years have now made a new phase of intensive and productive research pos-
sible within the field of immune defenses against infection. The aim of this
chapter on immunology, in a compact guide to medical microbiology, is to
present the immune system essentially as a system of defense against in-
fections and to identify its strengths and weaknesses to further our under-
standing of pathogenesis and prevention of disease.
The Immunological Apparatus
& The immune system is comprised of various continuously circulating cells
(T and B lymphocytes, and antigen-presenting cells present in various tis-
sues). T and B cells develop from a common stem cell type, then mature
in the thymus (T cells) or the bone marrow (B cells), which are called primary
(or central) lymphoid organs. An antigen-specific differentiation step then
takes places within the specialized and highly organized secondary (or per-
ipheral) lymphoid organs (lymph nodes, spleen, mucosa-associated lym-
phoid tissues [MALT]). The antigen-specific activation of B and/or T cells in-
volves their staggered interaction with other cells in a contact-dependent
manner and by soluble factors.
B cells bear antibodies on their surfaces (cell-bound B-cell receptors).
They secrete antibodies into the blood (soluble antibodies) or onto mucosal
surfaces once they have fully matured into plasma cells. Antibodies recognize
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46 2 Basic Principles of Immunology
the three-dimensional structures of complex, folded proteins, and hydro-
carbons. Chemically, B-cell receptors are globulins (“immunoglobulins”)
and comprise an astounding variety of specific types. Despite the division
of immunoglobulins into classes and subclasses, they all share essentially
2 the same structure. Switching from one Ig class to another generally requires
T-cell help.
T cells recognize peptides presented on the cell surface by major histo-
compatibility (gene) complex (MHC) molecules. A T-cell response can only
be initiated within organized lymphoid organs. Naive T cells circulate through
the blood, spleen, and other lymphoid tissues, but cannot leave these com-
partments to migrate through peripheral nonlymphoid tissues and organs
unless they are activated. Self antigens (autoantigens), presented in the thy-
mus and lympoid tissues by mobile lymphohematopoietic cells, induce T-cell
destruction (so-called negative selection). Antigens that are expressed only
in the periphery, that is outside of the thymus and secondary lymphoid or-
gans, are ignored by T cells; potentially autoreactive T cells are thus directed
against such self antigens. T cells react to peptides that penetrate into the
organized lymphoid tissues. New antigens are first localized within few lym-
phoid tissues before they can spread systemically. These must be present in
lymphoid tissues for three to five days in order to elicit an immune response.
An immune response can be induced against a previously ignored self antigen
that does not normally enter lymphoid tissues if its entry is induced by cir-
cumstance, for instance, because of cell destruction resulting from chronic
peripheral infection. It is important to remember that induction of a small
number of T cells will not suffice to provide immune protection against a
pathogen. Such protection necessitates a certain minimum sum of activated
T cells. &
The function of the immunological apparatus is based on a complex series
of interactions between humoral, cellular, specific, and nonspecific mechan-
isms. This can be better understood by examining how the individual com-
ponents of the immune response function.
The human immunological system can be conceived as a widely dis-
tributed organ comprising approximately 1012 individual cells, mainly lym-
phocytes, with a total weight of approximately 1 kg. Leukocytes arise from
pluripotent stem cells in the bone marrow, then differentiate further as two
distinct lineages. The myeloid lineage constitutes granulocytes and mono-
cytes, which perform important basic defense functions as phagocytes
(“scavenger cells”). The lymphoid lineage gives rise to the effector cells of
the specific immune response, T and B lymphocytes. These cells are con-
stantly being renewed (about 106 new lymphocytes are produced in every
minute) and destroyed in large numbers (see Fig. 2.17, p. 88). T and B lym-
phocytes, while morphologically similar, undergo distinct maturation pro-
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The Immunological Apparatus 47 2
cesses (Table 2.1, Fig. 2.2). The antigen-independent phase of lymphocyte
differentiation takes place in the so-called primary lymphoid organs:
T lymphocytes mature in the thymus and B lymphocytes in the bursa fabricI
(in birds). Although mammals have no bursa, the term B lymphocytes (or
B cells) has been retained to distinguish these cells, with their clearly distinct
functions and maturation in the bone marrow, from T lymphocytes, which
mature in the thymus (Table 2.1). B cells mature in the fetal liver as well
as in fetal and adult bone marrow. In addition to their divergent differentia-
Maturation of B and T cells
Primary (central) lymphoid organs Secondary (peripheral) lymphoid organs
Antigen-independent Antigen-dependent
Progenitor Precursor B Immature Mature Activated Blast Plasma cell
B (pro-B) cell (pre-B) cell B cell IgM B cell B cell IgD B cell µ
B cells µ µ
λ5/Vpre B1,2 λ or κ IgM IgM
IgD IgM
Bone marrow CD4 αβ
Stem cell CD4 CD4 αβ CD8 Mature T cells Effector T
(Te) cells
β ρTα β
CD8 αβ Activation in secondary
T cells Immature T cells ± selection
Thymic cortex lymphoid organs
(via contact and/
Thymic medulla or interleukins)
Fig. 2.2 All lymphoid cells originate from pluripotent stem cells present in the
bone marrow which can undergo differentiation into B or T cells. Stem cells
that remain in the bone marrow develop into mature B cells via several anti-
gen-independent stages; including the k5Vpre-B cell stage, and pre-B cells
with a special k5 precursor chain. Antigen contact within secondary lymphoid or-
gans can then activate these cells, finally causing them to differentiate into anti-
body-secreting plasma cells.
T cells mature in the thymus; pTa is a precursor a chain associated with TCRb chain
surface expression. The pTa chain is later replaced by the normal TCRa chain.
Immature CD4+ CD8+ double-positive thymocytes are localized within the cortical
region of the thymus; some autoreactive T cells are deleted in the cortex, whilst
some are deleted in the medulla as mature single-positive T cells. The remaining
T cells mature within the medulla to become CD4+ CD8– or CD4– CD8+ T cells.
From here, these single positive T cells can emigrate to peripheral secondary
lymphoid organs, where they may become activated by a combination of antigen
contacts, secondary signals, and cytokines.
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48 2 Basic Principles of Immunology
Table 2.1 Distribution of Lymphocyte Subpopulations and APCs in Various Or-
gans (% of All Mononuclear Cells)
2 Peripheral blood B T NK, LAK, ADCC APC
Lymph 10–15 70–80 5–10 <1
Thoracic duct 5 95–100 ? ?
Thymus 5–10 90–95 ? ?
Bone marrow 1 95–100 ? 0
Spleen 15–20 10–15 ? 0
Lymph nodes, tonsils, etc. 40–50 40–60 20–30 1
20–30 70–80 5–8 1
NK: natural killer cells; LAK: lymphokine-activated killer cells, ADCC: antibody-depen-
dent cellular toxicity, APC: antigen-presenting cells
tion pathways, T and B cells differ with respect to their functions, receptors,
and surface markers. They manifest contrasting response patterns to cyto-
kines, and display a marked preference to occupy different compartments
of lymphoid organs. T and B cells communicate with each other, and with
other cell types, by means of adhesion and accessory molecules (CD antigens,
see Table 2.13, p. 137) or in response to soluble factors, such as cytokines,
which bind to specific receptors and induce the activation of intracellular sig-
naling pathways. The antigen-dependent differentiation processes which
leads to T and B cell specialization, takes place within the secondary lym-
phoid organs where lymphocytes come into contact with antigens. As a
general rule the secondary lymphoid organs contain only mature T and B
cells, and comprise encapsulated organs such as the lymph nodes and
spleen, or non-encapsulated structures which contain lymphocytes and
are associated with the skin, mucosa, gut, or bronchus (i.e. SALT, MALT,
GALT, and BALT). Together, the primary and secondary lymphoid organs ac-
count for approximately 1–2 % of body weight.
The B-Cell System
& B lymphocytes produce antibodies in two forms; a membrane-bound
form and a secreted form. Membrane-bound antibody forms the B-cell anti-
gen receptor. Following antigen stimulation, B lymphocytes differentiate into
plasma cells, which secrete antibodies exhibiting the same antigen specifi-
city as the B-cell receptor. This system is characterized as humoral immu-
nity, due to this release of receptors into the “humoral” system which
constitutes vascular contents and mucous environments. The humoral
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The B-Cell System 49
Table 2.2 Characteristics of the Various Immunoglobulin Classes
IgM IgD IgG IgE IgA
Svedberg unit 19 S 7S 7S 8S 7 S, 9 S, 11 S 2
900 kDa 185 kDa 150 kDa 200 kDa 160 kDa
Molecular weight 5 1 1 1 1, 2, 3
l (4) d (3) c (3) e (4) a (3)
Number of dimeric units
ÀÀÀÀÀÀÀÀÀÀ j or k ÀÀÀÀÀÀÀÀÀÀ!
H chain 10 2 2 2 2, 4, 6
(constant domains)
L chain
Antigen-binding sites
(ABS)
Concentration in 0.5–2 0–0.4 8–16 0.02–0.50 1.4–4
normal serum (g/l)
6 0–1 80 0.002 13
% of Ig 1–2 ? 7–21
1–2 3–6
Half-life (days) in serum
>200 on
mast cells
Complement (C) activation:
Classic +–+––
Alternative ––––+
Placental passage – – + + –
Binding to mast cells – – – + –
and basophils
Binding to macrophages, – – (+) – (+)
granulocytes,
and thrombocytes
Subclasses – – + (4) – + (2)
IgG subclasses IgG1 IgG2 IgG3 IgG4
% of total IgG 60–70 14–20 4–8 2–6
Reaction to Staphlococcus protein A + + – +
Placental passage + (+) + +
Complement (C) activation: +++ ++ ++++ (+)
Binding to monocytes/macrophages +++ + +++ (+)
Blocks IgE binding (–) – – +
Half-life (days) 21–23 21–23 7–9 21–23
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50 2 Basic Principles of Immunology
system also contains non-specific defense mechanisms, including the com-
plement system (see “Immune response and effector mechanisms,” p. 66ff.).
In chemical terms, B-cell receptors are globulins (Ig or immunoglobulins).
These immunoglobulins comprise a number of classes and subclasses, as
2 well as numerous different specificities, but share a common structure
(Fig. 2.3a). &
Immunoglobulin Structure
All immunoglobulin monomers have the same basic configuration, in that
they consist of two identical light chains (L) and two identical heavy chains
(H). The light chains appear as two forms; lambda (k) or kappa (j). There are
five main heavy chain variants; l, d, c, a, and e. The five corresponding im-
munoglobulin classes are designated as IgM, IgD, IgG, IgA, or IgE, depending
on which type of heavy chain they use (Fig. 2.3b). A special characteristic of
the immunoglobulin classes IgA and IgM is that these comprise a basic
monomeric structure that can be doubled or quintupled (i.e., these can exist
in a dimeric or pentameric form). Table 2.2 shows the composition, mole-
cular weights and serum concentrations of the various immunoglobulin
classes (p. 49).
Fig. 2.3 a Immunoglobulin monomers. The upper half of the figure shows the
intact monomer consisting of two L and two H chains. The positions of the dis-
ulfide bonds, the variable N-terminal domains, and the antigen-binding site (ABS)
are indicated. The lower half of the figure shows the monomers of the individual
polypeptide chains as seen following exposure to reducing conditions (which break
the disulfide bonds) and denaturing conditions; note that the ABS is lost. Papain
digestion produces two monovalent Fab fragments, and one Fc fragment. Follow-
ing pepsin digestion (right), the Fc portion is fragmented, but the Fab fragments
remain held together by disulfide bonds. The F(ab’)2 arm is bivalent (with two
identical ABS). Fv fragments comprise a single-chain ABS formed by recombinant
technology. These consist of the variable domains of the H and L chains, joined
covalently by a synthetic linker peptide.
b Classes of immunoglobulins. IgM, IgD, IgG, IgA, and IgE are differentiated
by their respective heavy chains (l, d, c, a, e). IgA (a chain) forms dimers held
together by the J (joining) chain; the secretory (S) piece facilitates transport of
secretory IgA across epithelial cells, and impairs its enzymatic lysis within secre-
tions. IgM (l chain) forms pentamers with 10 identical ABS; the IgM monomers
are held together by J chains. The light chains (k and j) are found in all classes of
immunoglobulins. "
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The B-Cell System 51 2
Immunoglobulins contain numerous domains, as illustrated by the struc-
ture of IgG. In monomeric IgG each domain consists of a protein segment
which is approximately 110 amino acids in length. Both light chains possess
two such domains, and each heavy chain possesses four or five domains. The
domain structure was first revealed by comparison of the amino acid se-
quence derived from many different immunoglobulins belonging to the
Basic Immunoglobulin Structures ABS
Ig monomer
Fab Fab F(ab')2
Papain Pepsin Variable
domains
Splitting of Constant
disulfide bonds domains
Fc
H chain L chain Fv
L chain
a H chain
IgG λ or κ IgE
γ ε
IgM
IgA J chain
α λ oder κ
S piece J chain
λ or κ
µ
b
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52 2 Basic Principles of Immunology
Table 2.3 Antigen Recognition by B and T Cells
B lymphocytes T helper cells Cytotoxic T cells
(CD4+) (CTL; CD8+)
2 Recognition Surface Ig (BCR) TCR TCR
structure of B
or T cell
Recognized Conformational Linear epitopes only Linear epitopes (pep-
epitope epitopes (no MHC
restriction) (10–15 amino acids) + tides) (8)–9–(10)
MHC class II amino acids + MHC
class I
Antigen type Proteins/carbo- Peptides only Peptides only
hydrates
Antigen Not necessary Via MHC class II Via MHC class I
presentation structures structures
Effectors Antibodies Signals induced Cytotoxicity mediat-
(+/– complement) by contact ed by contact (per-
(T/B help) or forin, granzyme), or
cytokines release of cytokines
same class. In this way a high level of sequence variability was revealed to be
contained within the N-terminal domain (variable domain, V), whilst such
variability was comparably absent within the other domains (constant do-
mains, C). Each light chain consists of one variable domain (VL) and one con-
stant domain (CL). In contrast, the heavy chains are roughly 440–550 amino
acids in length, and consist of four to five domains. Again, the heavy chain
variable region is made up of one domain (VH), whereas the constant region
consists either of three domains (c, a, d chains), or four domains (l, e chains)
(CH1, CH2, CH3, and CH4). Disulfide bonds link the light chains to the heavy
chains and the heavy chains to one another. An additional disulfide bond
is found within each domain.
The three-dimensional form of the molecule forms a letter Y. The two
short arms of this ’Y’ consist of four domains each (VL, CL, VH, and CH1),
and this structure contains the antigen-binding fragments—hence its desig-
nation as Fab (fragment antigen binding). The schematic presented in Fig. 2.3
is somewhat misleading, since the two variable domains of the light and
heavy chains are in reality intertwined. The binding site—a decisive structure
for an epitope reaction—is formed by the combination of variable domains
from both chains. Since the two light chains, and the two heavy chains, con-
tain identical amino acid sequences (this includes the variable domains), each
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The B-Cell System 53 2
immunoglobulin monomer has two identical antigen-binding sites (ABS),
and these form the ends of the two short arms of the ’Y’. An area within
the antibody consisting of 12–15 amino acids contacts the peptide region
contained within the antigen and consisting of approximately 5–800 A˚ 2
(Table 2.3). The trunk of the ’Y’ is called the Fc fragment (named, “fraction
crystallizable” since it crystallizes readily) and is made up of the constant
domains of the heavy chains (CH2 and CH3, and sometimes CH4).
Diversity within the Variable Domains
of the Immunoglobulins
The specificity of an antibody is determined by the amino acid sequence of
the variable domains of the H and L chains, and this sequence is unique for
each corresponding cell clone. How has nature gone about the task of produ-
cing the needed diversity of specific amino acid sequences within a biochemi-
cally economical framework? The genetic variety contained within the B-cell
population is ensured by a process of continuous diversification of the geneti-
cally identical B-cell precursors. The three gene segments (variable, diversity,
joining) which encode the variable domain (the VDJ region for the H chain,
and the VJ region for the L chain) are capable of undergoing a process called
recombination. Each of these genetic segments are found as a number of var-
iants (Fig. 2.4, Table 2.4). B-cell maturation involves a process of genetic re-
Table 2.4 Organization of the Genetic Regions for the Human Immunoglobu-
lins and T-Cell Receptors (TCR)
Immunoglobulins TCRab TCRcd
HL ab cd
V segments 95 150 50–100 75–100 96
– 2 –3
D segments 23 – 60–80 13 53
VJ VD, DJ VJ VD
J segments 9 12
54
Nucleotide VD, DJ VJ
additions
Number of potential 15 000 8000
combinations for V
(H + L)
Theoretical upper limit >1012 >1012 >1012
of all combinations
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54 2 Basic Principles of Immunology
combination resulting in a rearrangement of these segments, such that
one VH, one DH, and one JH segment become combined. Thus the germ
line does not contain one gene governing the variable domain, but rather
gene segments which each encode fragments of the necessary information.
2 Mature B cells contain a functional gene which, as a result of the recombina-
tion process, is comprised of one VHDHJH segment. The diversity of T-cell
receptors is generated in a similar manner (see p. 57).
Fig. 2.4 explains the process of genetic recombination using examples of
an immunoglobulin H chain and T-cell receptor a chain.
The major factors governing immunoglobulin diversity include:
& Multiple V gene segments encoded in the germ lines.
& The process of VJ, and VDJ, genetic recombination.
& Combination of light and heavy chain protein structures.
& Random errors occurring during the recombination process, and inclusion
of additional nucleotides.
& Somatic point mutations.
In theory, the potential number of unique immunoglobulin structures that
could be generated by a combination of these processes exceeds 1012, how-
ever, the biologically viable and functional range of immunoglobulin specifi-
cities is likely to number closer to 104.
The Different Classes of Immunoglobulins
Class switching. The process of genetic recombination results in the genera-
tion of a functional VDJ gene located on the chromosome upstream of those
Fig. 2.4 a Heavy chain of human IgG. The designations for the gene segments
in the variable part of the H chain are V (variable), D (diversity), and J (joining).
The segments designated as l, d, c, a, and e code for the constant region
and determine the immunoglobulin class. The V segment occurs in several hun-
dred versions, the D segment in over a dozen, and the J segment in several forms.
V, D, and J segments combine randomly to form a sequence (VDJ) which codes for
the variable part of the H chain. This rearranged DNA is then transcribed, creating
the primary RNA transcript. The non-coding intervening sequences (introns) are
then spliced out, and the resulting mRNA is translated into the protein product.
b a chain of mouse T-cell receptor. Various different V, D, and J gene segments
(for b and d), V and J gene segments (for a and c) are available for the T-cell re-
ceptor chains. The DNA loci for the d chain genes are located between those for
the a chain. "
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The B-Cell System 55
Rearrangement of the B- and T-Cell Receptor Genes
2
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56 2 Basic Principles of Immunology
regions encoding the H chain segments Cl, Cd, Cc, Ca, and Ce, in consecutive
order. Thus all immunoglobulin production begins with the synthesis of IgM
and IgD (resulting from transcription of the VDJ and the Cl or Cd gene seg-
ments). This occurs without prior antigen stimulus and is transitional in nat-
2 ure. Antigen stimulation results in a second gene rearrangement—during
which the VDJ gene is relocated to the vicinity of Cc, Ca, or Ce by a process
of recombination involving deletion of the intervening regions. Following this
event, the B cell no longer produces H chains of the IgM or IgD classes, but is
instead committed to the production of IgG, IgA, or IgE—thus allowing secre-
tion of the entire range of immunoglobulin types (Table 2.2). This process is
known as class switching, and results in a change of the Ig class of an antibody
whilst allowing its antigen specificity to be retained.
Variability types. The use of different heavy or light chain constant regions
results in new immunoglobulin classes known as isotypes. Individual Ig
classes can also differ, with such genetically determined variations in the con-
stant elements of the immunoglobulins (which are transmitted according to
the Mendelian laws) are known as allotypes. Variation within the variable
region results in the formation of determinants, known as idiotypes. The
idiotype determines an immunoglobulins antigenic specificity, and is unique
for each individual B-cell clone.
Functions. Each different class of antibody has a specific set of functions. IgM
and IgD act as B-cell receptors in their earlier transmembrane forms, although
the function of IgD is not entirely clear. The first antibodies produced in the
primary immune response are IgM pentamers, the action of which is directed
largely against micro-organisms. IgM pentamers are incapable of crossing the
placental barrier. The immunoglobulin class which is most abundant in the
serum is IgG, with particularly high titers of this isotype being found following
secondary stimulation. IgG antibodies pass through the placenta and so pro-
vide the newborn with a passive form of protection against those pathogens
for which the mother exhibits immunity. In certain rare circumstances such
antibodies may also harm the child, for instance when they are directed
against epitopes expressed by the child’s own tissues which the mother
has reacted against immunologically (the most important clinical example
of this is rhesus factor incompatibility). High concentrations of IgA antibodies
are found in the intestinal tract and contents, saliva, bronchial and nasal se-
cretions, and milk—where they are strategically positioned to intercept infec-
tious pathogens (particularly commensals) (Fig. 2.5). IgE antibodies bind to
high-affinity Fce receptors present on basophilic granulocytes and mast cells.
Cross-linking of mast cell bound IgE antibodies by antigen results in cellular
degranulation and causes the release of highly active biogenic amines (his-
tamine, kinines). IgE antibodies are produced in large quantities following
parasitic infestations of the intestine, lung or skin, and play a significant
role in the local immune response raised against these pathogens.
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The Mucosa-Associated Lymphoid Tissue The T-Cell System 57
(MALT) Immune System and “Homing” 2
Fig. 2.5 Specialized APCs (M cells in the intestinal wall or pulmonary macro-
phages in the lung) take up antigens in mucosa and present them in the Peyer’s
patches or local lymph nodes. This probably enhances T cell-dependent activation
of IgA-producing B cells, which are preferentially recruited to the mucosal regions
(“homing”) via local adhesion molecules and antigen depots, resulting in a type of
geographic specificity within the immune response.
The T-Cell System
T-Cell Receptors (TCR) and Accessory Molecules
Like B cells, T cells have receptors that bind specifically to their steric counter-
parts on antigen epitopes. The diversity of T-cell receptors is also achieved by
means of genetic rearrangement of V, D, and J segments (Fig. 2.4b). However,
the T-cell receptor is never secreted, and instead remains membrane-bound.
Each T-cell receptor consists of two transmembrane chains, of either
the a and b forms, or the c and d forms (not to be confused with the heavy
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58 2 Basic Principles of Immunology
chains of Ig bearing the same designations). Both chains have two extracel-
lular domains, a transmembrane anchor element and a short intracellular ex-
tension. As for Ig, the terminal domains are variable in nature (i.e., Va and Vb),
and together they form the antigen binding site (see Fig. 2.9, p. 65). T-cell
2 receptors are associated with their so-called co-receptors—other mem-
brane-enclosed proteins expressed on the T cell surface—which include
the multiple-chain CD3 complex, and CD4 or CD8 molecules (depending
on the specific differentiation of the T cell). CD stands for “cluster of differ-
entiation” or “cluster determinant” and represents differentiation antigens
defined by clusters of monoclonal antibodies. (Table 2.13, p. 135f., provides
a summary of the most important CD antigens.)
T-Cell Specificity and the
Major Histocompatibility Complex (MHC)
T-cell receptors are unable to recognize free antigens. Instead the T-cell re-
ceptor can only recognize its specific epitope once the antigen has been
cleaved into shorter peptide fragments by the presenting cell. These frag-
ments must then be embedded within a specific molecular groove and pre-
sented to the T-cell receptor (a process known as MHC-restricted T-cell re-
cognition or MHC restriction). This “binding groove” is located on the MHC
molecule. The MHC encodes for the powerful histocompatibility or trans-
plantation antigens (also known in humans as HLA, human leukocyte antigen
molecules, Fig. 2.6).
The designation “MHC molecule” derives from the initial discovery of
the function of the complex as a cell surface structure, responsible for the
The MHC Gene Complex
Chromosome 6
HLA gene sequence
TNF BC AG
HSP70
B1 61 18 41
C2
C4A
CYP21
C4B
DRA
DRB
DQA
DQB
DPA
DPB
Allele variants
(approximate count)
38 8 19 14 69 1
Class II III I
Fig. 2.6 The human major histocompatibility gene complex (HLA genes) is lo-
cated on chromosome 6. There are three different classes of MHC molecules.
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The T-Cell System 59 2
immunological rejection of cell transfusions or tissue and organ transplants.
Its true function as a peptide-presenting molecule was not discovered until
the seventies, when its role became apparent whilst testing the specificity of
virus-specific cytotoxic T cells. During these experiments it was observed that
immune T cells were only able to destroy infected target cells if both cell types
were derived from the same patient or from mice with identical MHC mol-
ecules. The resulting conclusion was that a T-cell receptor not only recognizes
the corresponding amino acid structure of the presented peptide, but addi-
tionally recognizes certain parts of the MHC structure. It is now known that
this contact between MHC on the APC and the T-cell receptor is stabilized by
the co-receptors CD4 and CD8.
MHC classes. Molecules encoded by the MHC can be classified into three
groups according to their distribution on somatic cells, and the types of cells
by which they are recognized:
& MHC class I molecules. These molecules consist of a heavy a chain with
three Ig-like polymorphic domains (these are encoded by 100–1000 alleles,
with the a1 and a2 domains being much more polymorphic than the a3 do-
main) and a nonmembrane-bound (soluble) single-domain b2 microglobulin
(b2M, which is encoded by a relatively small number of alleles). The a chain
forms a groove that functions to present antigenic peptides (Fig. 2.7). Human
HLA-A, HLA-B, and HLA-C molecules are expressed in varying densities on all
somatic cells (the relative HLA densities for fibroblasts and hepatic cells, lym-
phocytes, or neurons are 1x, 100x and 0.1Â, respectively). Additional, non-
classical, class I antigens which exhibit a low degree of polymorphism are
also present on lymphohematopoietic cells and play a role in cellular differ-
entiation.
& MHC class II molecules. These are made up by two different polymorphic
transmembrane chains that consist of two domains each (a1 is highly poly-
morphic, whilst b1 is moderately polymorphic, and b2 is fairly constant).
These chains combine to form the antigen-presenting groove. Class II mole-
cules are largely restricted to lymphohematopoetic cells, antigen-presenting
cells (APC), macrophages, and so on. (see Fig. 2.9a, p. 65) In humans, but
not in mice, they are also found on some epithelial cells, neuroendocrine cells,
and T cells. The products of the three human gene regions HLA-DP, HLA-DQ
and HLA-DR can additionally form molecules representing combinations of
two loci—thus providing additional diversity for peptide presentation.
& MHC class III molecules. These molecules are not MHC antigens in the
classical sense, but are encoded within the MHC locus. These include com-
plement (C) components C4 and C2, cytokines (IL, TNF), heat shock protein 70
(hsp70), and other products important for peptide presentation.
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60 2 Basic Principles of Immunology
Protein Structure of MHC Class I Molecules
2 α1 α2
N
N
β2M N
C C
b
a α3
Fig. 2.7 MHC class I translation antigen: a lateral view, b vertical view. The pre-
senting peptide is shown in violet. The three domains of the heavy chain are a1, a2,
and a3. b2 microglobulin (b2M) functions as a light chain, and is not covalently
bound to the heavy chain.
Functions of MHC molecules. MHC class I and II molecules function mainly as
molecules capable of presenting peptides (Figs. 2.7–2.9). These two classes of
MHC molecules present two different types of antigens:
— Intracellular antigens; these are cleaved into peptides within the protea-
some and are usually associated with MHC class I molecules via the en-
dogenous antigen processing pathway (Fig. 2.8, left side).
— Antigens taken up from exogenous sources; these are processed into
peptides within phagolysosomes, and in most cases are then presented
on MHC class II molecules on the cell surface (Fig. 2.8, right side). Within
the phagolysosome, a fragment called the invariant chain (CLIP, class II-
inhibiting protein) is replaced by an antigen fragment. This CLIP fragment
normally blocks the antigen-binding site of the MHC class II dimer, thus
preventing its occupation by other intracellular peptides.
The presentation groove of MHC class I molecules is closed at both ends, and
only accommodates peptides of roughly 8–10 (usually 9) amino acids in
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The T-Cell System 61
Presentation of Endogenous and Exogenous Antigens
2
Fig. 2.8 Intracellularly synthesized endogenous antigen peptides (left side) are
bound to MHC class I molecules within the endoplasmic reticulum, fixed into
the groove by b2M, and presented on the cell surface. Antigens taken up from
exogenous sources (right) are cleaved into peptides within phagosomes. The pha-
gosome then merges with endosomes containing MHC class II molecules, the
binding site of which had been protected by the so-called CLIP fragment. These
two presentation pathways functionally separate MHC class I restricted CD8+ T cells
from MHC class II restricted CD4+ T cells.
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62 2 Basic Principles of Immunology
length. The groove of MHC class II molecules is open-ended, and can contain
peptides of 9–15 (usually 10–12) amino acids in length.
T cells can only recognize antigenic peptides in combination with either
MHC class I (which presents endogenous linear peptides, such as those de-
2 rived from viruses) or MHC class II (which present exogenous linear peptides,
such as those derived from bacterial toxins) (Table 2.3). In contrast to anti-
bodies that recognize soluble, complex, nonlinear, three-dimensional struc-
tures—T-cell recognition is restricted to changes on the surfaces of cells signaled
via MHC plus peptide.
T-cell specificity. T-cell recognition therefore involves two levels of specifi-
city: first, MHC presentation molecules bind peptides with a certain degree
of specificity as determined by the shape of the groove and the peptide an-
choring loci. Second, the MHC-peptide complex will only be recognized by
specific T-cell receptors (TCR) once a minimum degree of binding strength
has been obtained. For this reason diseases associated with the HLA complex
are determined largely by the quality of peptide presentation, but can also be
influenced by the available TCR repertoire.
The structure of the MHC groove therefore determines which, of all the
potentially recognizable, peptides will actually be presented as T-cell epi-
topes. Thus, the same peptides cannot function as T-cell epitopes in all indi-
viduals. Nonetheless, certain combinations of peptides and MHC are fre-
quently observed. For example, approximately 50 % of Caucasians carry the
HLA-A2 antigen, although this is sometimes found in a variant form.
Antigen-presenting cells (APC). APCs belong to the lymphohematopoietic
system. They attach peptides to MHC class II molecules for presentation to
T cells, and induce T-cell responses. The complex mechanisms involved in
this process have not yet been fully delineated. Stromal cells present in
the thymus and bone marrow (i.e., connective tissue cells, dendritic cells
and nurse cells in both thymus and bone marrow, plus epithelial cells in
the thymus) can also function as APCs. The following cell types function
as APCs in peripheral secondary lymphoid organs:
& Circulating monocytes.
& Sessile macrophages in tissues, microglia in the central nervous system.
& Bone marrow derived dendritic cells with migratory potential—these oc-
cur as cutaneous Langerhans cells, as veiled cells during antigen transfer into
the afferent lymph vessels, as interdigitating cells in the spleen and lymph
nodes, and as interstitial dendritic cells or as M cells within MALT.
& Follicular dendritic cells (FDC)—these are found within the germinal cen-
ters of the secondary lymph organs, do not originate in the bone marrow, and
do not process antigens but rather bind antigen-antibody complexes via Fc
receptors and complement (C3) receptors.
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The T-Cell System 63 2
& B lymphocytes—these serve as a type of APC for T helper cells during T-B
collaborations.
The consequences of MHC variation. Because every individual differs with
regard to the set of polymorphic MHC molecules and self antigens expressed
(with the exception of monozygotic twins and inbred mice of the same strain),
the differences between two given individuals are considerable. The high de-
gree of variability in MHC molecules—essential for the presentation of a large
proportion of possible antigenic peptides for T-cell recognition—results in
these molecules becoming targets for T cell recognition following cellular
or organ transplantation resulting in transplant rejection. The term “trans-
plantation antigens” is therefore a misnomer, and is only used because their
real function was not discovered until a later time. Normally antigens are only
recognized by T cells if they are associated with MHC-encoded self-structures.
Transplant recognition, which apparently involves the imitation of the com-
bination of a non-self antigen plus a self-MHC molecule, can therefore be con-
sidered an exception. The process probably arises from T-cell receptor cross-
reactivity between host self-MHC antigens plus foreign peptides on the one
hand, and non-self transplantation antigens associated with self-peptides
from the donor on the other hand (for example, the T-cell receptor for
HLA-A2 peptide X cross-reacts with HLA-A13 peptide Y). Transplant rejection
is therefore a consequence of the enormous variety of combinations of anti-
genic peptide plus MHC, which is exhibited by each individual organism.
T-Cell Maturation: Positive and Negative Selection
Maturation of T cells occurs largely within the thymus. Fig. 2.2 (p. 47) shows a
schematic presentation of this process. Because the MHC-encoded presenta-
tion molecules are highly polymorphic, and are also subject to mutation, the
repertoire of TCRs is not genetically pre-determined. One prerequisite for
an optimal repertoire of T-cells is therefore the positive selection of T cells
such that these preferentially recognize peptides associated only with self
transplantation (MHC) antigens. A second prerequisite is negative selection,
which involves the deletion of T cells that react too strongly against self MHC
plus self peptide. The random processes governing the genetic generation of
an array of T-cell receptors results ab or cd receptor chain combinations
which are in the majority of cases are non-functional. Those T cells preserved
through to maturity represent cells carrying receptors capable of effectively
recognizing self-MHC molecules (positive selection). However, the T cells
within this group which express too high an affinity for self-MHC plus
self-peptides are deleted (negative selection).
The process of positive selection was demonstrated in experimental mice
expressing MHC class I molecules of type b (MHC classIb) from which the
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64 2 Basic Principles of Immunology
thymus had been removed (and which therefore had no T cells). Implantation
of a new thymus with MHC class I molecules of type a (MHC class Ia) into the
MHC class Ib mice resulted in the maturation of T cells which only recognized
peptides presented by MHC class Ia molecules, and not peptides presented by
2 MHC class Ib molecules. However, recent experiments have shown that this is
probable an experimental artefact and that it is not (or not solely) the thymic
epithelial cells that determine the selection process, but that this process is
driven by cells formed in the bone marrow. Positive selection is generally
achieved by weak levels of binding affinity between the T-cell receptor
and the self-MHC molecules, whereas negative selection eliminates those
T cells exhibiting the highest levels of affinity (namely the self-or auto-reac-
tive T cells) and absence of binding causes death by neglect. Thus, only T cells
with moderate binding affinities are allowed to mature and exit the thymus.
These T cells can potentially react to non-self (foreign) peptides presented by
self MHC molecules. The enormous proliferation of immature thymocytes is
paralleled by continuous cell death of large numbers of thymocytes (apop-
tosis, see summary in Fig. 2.17, p. 88). In general, the maturation and survival
of lymphocytes is considered to be dependent on a continuous, repetitive,
signaling via transmembrane molecules, and cessation of these signals is
usually taken as a reliable indicator of cell death.
T-Cell Subpopulations
In order to recognize the presented antigen, T cells require the specific T-cell
receptor and a molecule which functions to recognize the appropriate MHC
molecules (i.e. CD4 or CD8 which recognize MHC class II and MHC class I,
respectively). Thus T cells are classified into different subpopulations based
on the CD4 or CD8 surface molecules:
CD4+ T cells. These T cells recognize only MHC class II-associated antigens.
They are also called T helper cells due to their important role in T-B cell col-
laboration (Fig. 2.9a), although they exhibit many other additional functions.
CD4+ cells can produce, or induce, the production of cytokines by which
means they can activate macrophages and exercise a regulatory effect on
other lymphocytes (see p. 75f.). Although these cells sometimes demonstrate
an ability to cause cytotoxic destruction in vitro, this does not hold true in
vivo.
CD8+ T cells. Only MHC class I-associated antigens are recognized by the
CD8+ molecule. These cells are also known as cytotoxic T cells due to their
ability to destroy histocompatible virus-infected, or otherwise altered, target
cells as well as allogeneic cells. This effect can be observed both in vitro and
in vivo (Fig. 2.9b). Costimulatory molecules are not required for this lytic
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The T-Cell System 65
Interactions in T-Cell Antigen Recognition
Antigen-presenting cell or B cell Antigen-presenting cell or target cell
CD40 MHC V-CAM LFA-1 MHC B-7 2
LFA-1 class II B-7 class I
molecule (CD11a/18) molecule (CD80)
(CD11a/18) (CD80)
LFA-3
LFA-3
(CD58)
(CD58)
α2 β2 α3 β2M
α1 β1 α2 α1
Vα Vβ
Vα Vβ S
Cα Cβ
Cα Cβ
CD40L CD4 CD28 CD8
CD2
CD2 TCR–CD3 (naive) ICAM-1 TCR–CD3 CD28
ICAM-1 complex complex (naive)
VLA-4 (CD54)
(CD54) (CD49/29)
CD44
(act.)
CD44 T helper cell (CD4+) Cytotoxic T cell (CD8+)
ab
Fig. 2.9 a The interactions of APCs or B cells with CD4+ T cells (T helper cells) are
mediated by MHC class II molecules (heterodimers). b Interactions between CD8+
T cells (cytotoxic T cells) and their target cells are mediated by MHC class I mo-
lecules. The presenting peptide is shown in violet. “S” indicates a superantigen,
named after its capacity to activate many different T helper cells through its ability
to bind to the constant regions of both the MHC and TCR molecules (naive = non-
activated T cells, act. = activated T cells).
effector function. However, cytotoxicity is only one of several important func-
tions expressed by CD8+ T cells. They also have many other non-lytic func-
tions which they execute via the production, or induction of, cytokine release.
The designation (CD8+) T suppressor cell is misleading and should not be
used. It was originally coined to distinguish these cells from the function
of T helper cells, mentioned above. However, plausible documentation of a
suppressor effect by CD8+ T cells has only been obtained in a very small
number of cases. In most cases, this suppressive effect can in fact be explained
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66 2 Basic Principles of Immunology
by the direct elimination of APC (i.e., by changing the antigen kinetics), or
indirectly via cytokine effects (see Fig. 2.14, p. 78). Thus, the name suppressor
T cell suggests a regulatory function that in reality is unlikely to exist.
In general, more neutral names, such as CD4+ T cells or CD8+ T cells, are pre-
2 ferable. Whereas the cytotoxic effector cells in the spleen and lymph nodes
possess a heterodimeric (a + b chain) CD8+ T molecule, the function of CD8+ T
cells found in the intestinal wall and expressing the a-homodimeric CD8
molecule remains unclear.
cd T cells. As for the homologous ab heterodimer, the cd T-cell receptor is
associated with the CD3 complex within the cell membrane. The genetic se-
quence for the c and d chains resembles that of the a and b chains, however,
there are a few notable differences. The gene complex encoding the d chain is
located entirely within the V and J segments of the a chain complex. As a
result, any rearrangement of the a chain genes deletes the d chain genes.
There are also far fewer V segments for the c and d genes than for the a
and b chains. It is possible that the increased binding variability of the d
chains makes up for the small number of V segments, as a result nearly
the entire variability potential of the cd receptor is concentrated within
the binding region (Table 2.4, p. 53). The amino acids coded within this region
are presumed to form the center of the binding site.
T cells with cd receptors recognize certain class I-like gene products in as-
sociation with phospholipids and phosphoglycolipids. In peripheral lymphoid
tissues, only a small number of T cells express the cd and CD3 co-receptor,
however, many of the T cells found within the mucosa and submucosa ex-
press cd receptors.
cd T cells can be negative for CD4+ and CD8+, or express two a chains (but
no b chain) of the CD8+ molecule. Although it is assumed that cd T cells may
be responsible for early, low-specificity, immune defense at the skin and mu-
cosa, their specificities and effector functions are still largely unknown.
Immune Responses and Effector Mechanisms
& The effector functions of the immune system comprise antibodies and
complement-dependent mechanisms within body fluids and the mucosa,
as well as tissue-bound effector mechanisms executed by T cells and mono-
cytes/macrophages. B cells are characterized by antigen specificity. Following
antigen stimulation, specific B cells proliferate and differentiate into plasma
cells that secrete antibodies into the surroundings. The type of B-cell re-
sponse induced is determined by the amount and type of bound antigen
recognized. Induction of an IgM response in response to antigens which
are lipopolysaccharides—or which exhibit an highly organized, crystal-like
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Immune Responses and Effector Mechanisms 67
structure containing identical and repetitively arranged determinants—is a
highly efficient and T cell-independent process which involves direct
cross-linking of the B-cell receptor. In contrast to this process, antibody re-
sponses against monomeric or oligomeric antigens are less efficient and
strictly require T cell help, for both non-self and self antigens. 2
Some forms of T-cell responses involve the release of soluble mediators
(cytokines), which effectively expands the field of T cell function beyond in-
dividual cell-to-cell contacts to an ability to regulate the function of large
numbers of surrounding cells. Other T-cell effector mechanisms are mediated
in a more precise manner through cell-to-cell contacts. Examples of this in-
clude perforin-dependent cytolysis and induction of the signaling pathways
involved in B-cell differentiation or Ig class switching. &
B Cells
B-Cell Epitopes and B-Cell Proliferation
Burnet’s clonal selection theory, formulated in 1957, states that every B-cell
clone is characterized by an unique antigen specificity, i.e., it bears a specific
antigen receptor. Accordingly, once rearrangement of the Ig genes has taken
place, the corresponding protein will be expressed as a surface receptor. At
the same time further rearrangement is stopped. Thus, only one ABS, or one
specificity (one VH plus VL [either j or k]), derived from a single allele can be
expressed on a single cell. This phenomenon is called allelic exclusion. The
body faces a large number of different antigens in its lifetime, necessitating
that a correspondingly large number of different receptor specificities, and
therefore different B cells, must continuously be produced. When a given
antigen enters an organism, it binds to the B cell which exhibits the correct
receptor specificity for that antigen. One way to describe this process is to say
that the antigen selects the corresponding B-cell type to which it most effi-
ciently binds. However, as long as the responding B cells do not proliferate,
the specificity of the response is restricted to a very small number of cells. For
an effective response, clonal proliferation of the responsive B cells must be
induced. After several cell divisions B cells differentiate into plasma cells
which release the specific receptors into the surroundings in the form of
soluble antibodies. B-cell stimulation proceeds with, or without, T cell
help depending on the structure and amount of bound antigen.
Antigens. Antigens can be divided into two categories; those which stimulate
B cells to secrete antibodies without any T-cell help, and those which require
additional T-cell signals for this purpose.
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68 2 Basic Principles of Immunology
& Type 1 T-independent antigens (TI1). These include paracrystalline,
identical epitopes arranged at approximately 5–10 nm intervals in a repetitive
two-dimensional pattern (e.g., proteins found on the surface of viruses, bac-
teria, and parasites); and antigens associated with lipopolysaccharides (LPS).
2 Thus TI1 antigens represent structures with a repetitive arrangement, which
allows the engagement of several antigen receptors at one time and results in
optimal Ig receptor cross-linking; or structures which result in sub-optimal
cross-linking, but which are complemented by an LPS-mediated activation
signal. Either type of antigen can induce B cell activation in the absence of
T cell help.
& Type 2 T-independent antigens (TI2). These antigens are less stringently
arranged, and are usually flexible or mobile on cell surfaces. They can cross-
link Ig receptors, but to a lesser extent than TI1 antigens. TI2 antigens require
a small amount of indirectly associated T help in order to elicit a B-cell re-
sponse (e.g., hapten-Ficoll antigens or viral glycoproteins on infected cell sur-
faces).
& T help-dependent antigens. These are monomeric or oligomeric (usually
soluble) antigens that do not cause Ig cross-linking, and are unable to induce
B-cell proliferation on their own. In this case an additional signal, provided by
contact with T cells, is required for B-cell activation (see also B-cell tolerance,
p. 93ff.).
Receptors on the surface of B cells and soluble serum antibodies usually re-
cognize epitopes present on the surface of native antigens. For protein anti-
gens, the segments of polypeptide chains involved are usually spaced far
apart when the protein is in a denatured, unfolded, state. A conformational
or structural epitope is not formed unless the antigen is present in its native
configuration. So-called sequential or linear epitopes—formed by contigu-
ous segments of a polypeptide chain and hidden inside the antigen—are lar-
gely inaccessible to B cell receptors or antibodies, as long as the antigen mol-
ecule or infectious agent retains its native configuration. These epitopes
therefore contribute little to biological protection. The specific role of linear
epitopes is addressed below in the context of T cell-mediated immunity. B
cells are also frequently found to be capable of specific recognition of sugar
molecules on the surface of infectious agents, whilst T cells appear to be in-
capable of recognizing such sugar molecules.
Proliferation of B cells. As mentioned above, contact between one, or a few,
B-cell receptors and the correlating antigenic epitope does not in itself suffice
for the induction of B-cell proliferation. Instead proliferation requires either a
high degree of B cell receptor cross-linking by antigen, or additional T cell-
mediated signals.
Proliferation and the rearrangement of genetic material—a continuous
process which can increase cellular numbers by a million-fold—occasionally
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Immune Responses and Effector Mechanisms 69 2
result in errors, or even the activation of oncogenes. The results of this process
may therefore include the generation of B-cell lymphomas and leukemia’s.
Since the original error occurs in a single cell, such tumors are monoclonal.
Uncontrolled proliferation of differentiated B cells (plasma cells) results in
the generation of monoclonal plasma cell tumors known as multiple mye-
lomas or plasmocytomas. Occasionally, myelomas produce excessive
amounts of the light chains of the monoclonal immunoglobulin, and these
proteins can then be detected in the urine as Bence-Jones proteins. Such
proteins represented some of the first immunoglobulin components acces-
sible for chemical analysis and they revealed important early details regard-
ing immunoglobulin structure.
Monoclonal Antibodies
A normal immune response usually involves the response and proliferation of
numerous B cell clones, bearing ABS with varying degrees of specificity for
the different epitopes contained within the antigen. Thus the immune re-
sponse is normally polyclonal. It is possible to isolate a single cell from
such a polyclonal immune response in an experimental setting. Fusing
this cell with an “immortal” proliferating myeloma cell results in generation
of a hybridoma, which then produces chemically uniform immunoglobulins
of the original specificity, and in whatever amounts are required. This method
was developed by Koeler and Milstein in 1975, and is used to produce mono-
clonal antibodies (Fig. 2.10), which represent important tools for experimen-
tal immunology, diagnostics, and therapeutics. Many monoclonal antibodies
are still produced in mouse and rat cells, making them xenogeneic for hu-
mans. Attempts to avoid the resulting rejection problems have involved
the production of antibodies by human cells (which remains difficult), or
the “humanization” of murine antibodies by recombinant insertion of the
variable domains of a murine antibody adjacent to the constant domains
of a human antibody. The generation of a transgenic mice, in which the Ig
genes have been replaced by human genes, has made the production of hy-
bridoma’s producing completely human antibodies possible.
T-Independent B Cell Responses
B cells recognize antigens via the Ig receptor. However, if the antigen is in a
monomeric, or oligomeric, soluble form the B cell can only mount a response
if it undergoes the process of T-B collaboration. Many infectious pathogens
carry surface antigens with polyclonal activation properties (e.g., lipopoly-
saccharide [LPS]) and/or crystal-like identical determinants, which are often
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70 2 Basic Principles of Immunology
Production of Monoclonal Antibodies
2
Fig. 2.10 Monoclonal antibodies are produced with the help of cell lines obtained
from the fusion of a B lymphocyte to an immortal myeloma cell. In the first in-
stance, mice are immunized against an antigen. They then receive a second, in-
travenous, dose of antigen two to four days before cell fusion. Then spleen cells are
removed and fused to the myeloma cell line using polyethylene glycol (PEG). Those
spleen cells that fail to fuse to a myeloma cell die within one day of culture. Next,
the fused cells are subjected to selection using HAT medium (hypoxanthine, ami-
nopterin, thymidine). Aminopterin blocks specific metabolic processes, but with
the help of the intermediary metabolites (hypoxanthine and thymidine) spleen
cells are able to complete these processes using auxiliary pathways. The myeloma
cells, on the other hand, have a metabolic defect which prevents them from utiliz-
ing such alternative pathways and resulting in the death of those cells cultured in
HAT medium. However, once a spleen cell has fused with a myeloma cell, the fused
spleen-myeloma product (hybridoma) is HAT-resistant. In this way only the suc-
cessfully fused cells will be able to survive several days of culture on HAT medium.
After this time, the cell culture is diluted such that there is, ideally, only one hy-
bridoma within each well. Individual wells are then tested for the presence of the
desired antibody. If the result is positive, the hybridoma cells are subcloned several
times to ensure clonality; with the specificity of the produced antibody being
checked following each round to subcloning. Production of purely human mono-
clonal antibodies is carried out using mice whose Ig genes have been completely
replaced by human Ig genes.
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Immune Responses and Effector Mechanisms 71 2
repeated in a regular pattern (linear e.g., flagella, or two-dimensional e.g.,
viruses) with intervals of 5–10 nm. These paracrystalline-patterned antigens
are capable of inducing B-cell responses without contact-dependent T cell
help. This probably occurs by means of maximum Ig receptor cross-linking.
Such B-cell responses are usually of the IgM type, since switching to different
isotype classes is either impossible or very inefficient in the absence of T cell
help. The IgM response is of a relatively brief duration (exhibiting a half-life of
about 24 h), but can nonetheless be highly efficient. Examples of this effi-
ciency include IgM responses induced by many viral envelope antigens which
bear neutralizing (“protective”) determinants accessible to the corresponding
antibodies, and responses to bacterial surface antigens (e.g., flagellae, lipo-
polysaccharides) or parasites.
T Cells
T-Cell Activation
There are two classes of T cells; T helper cells (CD4+) and cytotoxic T cells
(CD8+). Table 2.5 summarizes the reliance of T-cell responses on the dose,
localization, and duration of presence of antigen. T-cell stimulation via the
TCR, accessory molecules and adhesion molecules results in the activation
Table 2.5 Dependence of T-Cell Response on Antigen Localization, Amount,
and Duration of Presence
Antigen T-cell response
Localization Amount Duration of Negative selection by
Thymus presence deletion
Small-large Always
Blood, spleen, Small Short (1 day) No induction
lymph nodes Small Long (7 days) Induction
(secondary
lymphoid Large Short No induction
organs) Large Long (>10 days) Exhaustive induction/
deletion (anergy?)
Peripheral non Large or small Always or short Ignorance, indifference
lymphoid tissue
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72 2 Basic Principles of Immunology
of various tyrosine kinases (Fig. 2.11) and mediates stringent and differential
regulation of several signaling steps. T-cell induction and activation result
from the activation of two signals. In addition to TCR activation (signal 1 =
antigen), a costimulatory signal (signal 2) is usually required. Important cost-
2 imulatory signals are delivered by the binding of B7 (B7.1 and B7.2) proteins
(present on the APC or B cell) to ligands on the Tcells (CD28 protein, CTLA-4), or
by CD40–CD40 ligand interactions. T-cell expansion is also enhanced by IL-2.
T-Cell Activation by Superantigens
In association with MHC class II molecules, a number of bacterial and possibly
viral products can efficiently stimulate a large repertoire of CD4+ T cells at one
time. This is often mediated by the binding of the bacterial or viral product
to the constant segment of certain Vb chains (and possibly Va chains) with a
low level of specificity (see Fig. 2.9a, p. 65). Superantigens are categorized as
either exogenous or endogenous. Exogenous superantigens mainly include
bacterial toxins (staphylococcus enterotoxin types A-E [SEA, SEB, etc.]), toxic
shock syndrome toxin (TSST), toxins from Streptococcus pyogenes, and certain
retroviruses. Endogenous superantigens are derived from components of
certain retroviruses found in mice, and which display superantigen-like be-
havior (e.g., murine mammary tumor virus, MMTV). The function of super-
antigens during T cell activation can be compared to the effect of bacterial
lipopolysaccharides on B cells, in that LPS-induced B cell activation is also
polyclonal (although it functions by way of the LPS receptors instead of
the Ig receptors (see below)).
Interactions between Cells of the Immune System
T Helper Cells (CD4+ T Cells) and
T-B Cell Collaboration
Mature T cells expressing CD4 are called T helper (Th) cells (see also p. 64f.),
reflecting their role in co-operating with B cells. Foreign antigens, whose
three-dimensional structures are recognized by B cells, also contain linear
peptides. During the initial phase of the T helper cell response, these anti-
gens are taken up by APCs, processed, and presented as peptides in associa-
tion with MHC class II molecules—allowing their recognition by Th cells (see
Fig. 2.8, p. 61 and Fig. 2.13, p. 76). Prior to our understanding of MHC restric-
tion, B-cell epitopes were known as haptens, whilst those parts of the
antigens which bore the T-cell epitope were known as carriers. In order
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Immune Responses and Effector Mechanisms 73
T-Cell Activation
2
Fig. 2.11 Regulation of T-cell activation is controlled by multiple signals, including
costimulatory signals (Signal 2). Stimulation of the T cell via the T-cell receptor
(TCR; Signal 1) activates a tyrosine kinase, which in turn activates phospholipase
C (PLC). PLC splits phosphatidylinositol bisphosphate (PIP2) into inositol trispho-
sphate (IP3) and diacyl glycerol (DAG). IP3 releases Ca2+ from intracellular depots,
whilst DAG activates protein kinase C (PKC). Together, Ca2+ and PKC induce and
activate the phosphoproteins required for IL-2 gene transcription within the cell
nucleus. Stimulation of a T cell via the TCR alone results in production of only
very small amounts of IL-2. Increased IL-2 production often requires additional sig-
nals (costimulation, e.g., via CD28). Costimulation via CD28 activates tyrosine ki-
nases, which both sustain the transcription process and ensure post-transcriptional
stabilization of IL-2 mRNA. Immunosuppressive substances (in red letters) include
cytostatic drugs, anti-TCR, anti-CD3, anti-CD28 (CTLA4), anti-CD40, cyclosporine
A and FK506 (which interferes with immunophilin-calcineurin binding, thus reduc-
ing IL-2 production), and rapamycin (which binds to, and blocks, immunophilin
and hardly reduces IL-2 at all). Anti-interleukins (especially anti-IL-2, or a combina-
tion of anti-IL-2 receptor and anti-IL-15) block T-cell proliferation.
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